applications > All applications
287
NanoBRET™ assay for monitoring of ligand binding to GPCRs in live cells, using the CLARIOstar® and the PHERAstar FS

Leigh A. Stoddart(1), Elizabeth K. M. Johnstone(2,3), Amanda J. Wheal(1), Joëlle Goulding(1), Matthew B. Robers(4), Thomas Machleidt(4), Giovanni Abbenante(5), Keith V. Wood(4), Stephen J. Hill(1-3), and Kevin D. G. Pfleger(2,3)
1 Cell Signalling Research Group, School of Life Sciences, The University of Nottingham Medical School, Nottingham, United Kingdom.
2 Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, Nedlands, Western Australia, Australia.
3 Centre for Medical Research, The University of Western Australia, Crawley, Western Australia, Australia.
4 Promega Corporation, Madison, Wisconsin, United States.
5 BMG LABTECH Pty Ltd, Mornington, Victoria, Australia., 09/2015

A number of assay techniques are currently used to study ligand binding to GPCRs in living cells. The most well-known method involves radioactively labelled ligands which, unfortunately, is labor intensive, needs dedicated equipment, and raises disposal and safety issues. More recently, FRET (Fluorescence Resonance Energy Transfer) and Time Resolved-FRET techniques have been introduced. However, measuring fluorescence always requires an excitation source, such as a high energy lamp or a laser. As cells display substantial auto-fluorescence, sample excitation often leads to high background signals. TR-FRET addresses this issue, but requires chemically based lanthanide labeling of receptors.

In contrast to FRET, BRET (Bioluminescence Resonance Energy Transfer) does only need the addition of a suitable substrate to generate light/energy which then excites the fluorescent binding partner. The BRET donor, a luciferase, can be expressed on the receptor of interest using an appropriate transfected cell line.

This application note describes the first use of the BRET method for monitoring ligand binding to GPCRs in live cells using the NanoLuc luciferase.